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PubMed2006

Physical map-assisted whole-genome shotgun sequence assemblies.

We describe a targeted approach to improve the contiguity of whole-genome shotgun sequence (WGS) assemblies at run-time, using information from Bacterial Artificial Chromosome (BAC)-based physical maps. Clone sizes and overlaps derived from clone fingerprints are used for the calculation of length constraints between any two BAC neighbors sharing 40% of their size. These constraints are used to promote the linkage and guide the arrangement of sequence contigs within a sequence scaffold at the layout phase of WGS assemblies. This process is facilitated by FASSI, a stand-alone application that calculates BAC end and BAC overlap length constraints from clone fingerprint map contigs created by the FPC package. FASSI is designed to work with the assembly tool PCAP, but its output can be formatted to work with other WGS assembly algorithms able to use length constraints for individual clones. The FASSI method is simple to implement, potentially cost-effective, and has resulted in the increase of scaffold contiguity for both the Drosophila melanogaster and Cryptococcus gattii genomes when compared to a control assembly without map-derived constraints. A 6.5-fold coverage draft DNA sequence of the Pan troglodytes (chimpanzee) genome was assembled using map-derived constraints and resulted in a 26.1% increase in scaffold contiguity.

Animals↗
PubMed1999

Island length distribution in genome sequencing.

We consider the general problem of constructing a physical map of a genome by welding islands of overlapping clones. Both distribution of clone length and non-uniform probability of overlap detection are taken into account, the latter restricted to the Markov case in which only the location of the end of the developing island is required. Exact results for the distribution of island length are obtained in the special cases of fixed clone length or rigid overlap criterion, and mean and variance for the general situation. Determination of ocean length distribution permits island number and contig number distributions to be found as well.

Chromosome Mapping↗
PubMed1996

An ordered yeast artificial chromosome library covering over half of rice chromosome 6.

Yeast artificial chromosome (YAC) clones carrying DNA marker sequences located on the rice genetic map of chromosome 6 were ordered for physical mapping. A total of 122 restriction fragment length polymorphism markers, 16 sequence-tagged site markers, and five random amplified polymorphic DNA markers located, on average, at 0.9-cM intervals, were used for YAC clone screening by colony/Southern hybridization and PCR screening, respectively. A total of 216 individual YACs were selected from our YAC library of 7000 clones covering six genome equivalents. Each DNA marker could select, on average, 4.8 YAC clones, with 11 clones being the maximum. The YACs localized to the corresponding linkage map positions form 43 contigs and encompass about 60% of rice chromosome 6. This is the first step in constructing a physical map covering the whole rice genome by chromosome landing with YAC clones. These YACs and data will be used soon to isolate phenotypical trait genes by map-based cloning.

Blotting, Southern↗
PubMed1994

Isolation of two contigs of overlapping cosmids derived from human chromosomal band 3p21.1 and identification of 5 new 3p21.1 genes.

Consistent loss of DNA sequences from several regions on the short arm of human chromosome 3 has suggested that multiple tumor suppressor genes reside on chromosome 3p in various types of cancer cells. We have focused our efforts on an analysis of chromosomal band 3p21.1 since aminoacylase-1 (ACY1), which is localized to this band, has been shown to have lower levels of expression in several small cell and non-small cell lung cancer cell lines. Starting with two cosmids within 3p21.1, D3S92 and D3S93, we have isolated two separate contigs of overlapping cosmids within 3p21.1, by screening a library of 5700 chromosome 3-specific cosmid clones. Detailed restriction maps for these two contigs show that they contain multiple clusters of rare cutting restriction endonuclease sites. One contig extends for 100 kb and encompassed both ACY1 and D3S92, and the other extends about 80 kb around the D3S93 locus. Many different restriction fragments derived from these two contigs were found to be evolutionarily conserved and hybridized to distinct message transcripts. These fragments were used to identify homologous cDNAs from an adenogastric cDNA library, and several of these cDNAs were partially sequenced. We have identified five new genes from these two contigs and there is evidence to suggest that several additional genes reside within these cosmid contigs. The genes identified from 3p21.1 were then hybridized to DNA, isolated from a series of lung cancer cell lines and matched normal and tumor DNA from lung cancer patients. No alterations were detected with any of these probes, both at the DNA or RNA levels. A similar analysis with DNA fragments derived from these two genomic regions also failed to detect any alterations.

Animals↗
PubMed1996

A 350-kb cosmid contig in 3p14.2 that crosses the t(3;8) hereditary renal cell carcinoma translocation breakpoint and 17 aphidicolin-induced FRA3B breakpoints.

The constitutive fragile site at human chromosomal band 3p14.2, FRA3B, has been described as the most active common fragile site in the human genome. FRA3B is cytologically indistinguishable from the chromosome 3 breakpoint observed in the hereditary renal cell carcinoma (hRCC) translocation t(3;8) (p14.2;q24.13). Previous work demonstrated that a 1330-kb YAC clone, YC850A6, spans both the t(3;8) translocation and FRA3B and also encompasses FRA3B-associated breakpoints induced in hamster-human hybrids. This YAC was used to construct a multi-hit cosmid library. Screening of this library resulted in a 350-kb cosmid contig that extends distally from the t(3;8) translocation breakpoint. Seventeen aphidicolin-induced 3p14. 2 breakpoints derived from hamster-human hybrids were mapped within this cosmid contig. These breakpoints were found to localize as two distinct clusters, separated by 200 kb, which lie on either side of a region of frequent breakage within FRA3B as defined by FISH analysis using cosmids from the contigs. The most proximal of the breakpoint clusters lies approximately 100 kb distal to the hRCC t(3;8) breakpoint. The distribution of these breakpoints, together with the region of frequent chromosomal breakage mapped by FISH analysis, further confirms the position of FRA3B and helps to define the extent over which its fragility is exerted. These data indicate that FRA3B comprises several hundred kilobases of DNA sequence within 3p14.2. The 350-kb contig and the cosmid library constructed from YAC YC850A6 will be essential for further characterization of the region surrounding FRA3B and in experiments to determine the molecular basis of the fragility of FRA3B.

Animals↗
PubMed1999

Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments.

A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.

Animals↗
PubMed2001

Physical mapping across an avirulence locus of Phytophthora infestans using a highly representative, large-insert bacterial artificial chromosome library.

The oomycete plant pathogen Phytophthora infestans is the causal agent of late blight, one of the most devastating diseases of potato worldwide. As part of efforts to clone avirulence (Avr) genes and pathogenicity factors from P. infestans, we have constructed a bacterial artificial chromosome (BAC) library from an isolate containing six Avr genes. The BAC library comprises clones with an average insert size of 98 kb and represents an estimated 10 genome equivalents. A three-dimensional pooling strategy was developed to screen the BAC library for amplified fragment length polymorphism (AFLP) markers, as this type of marker has been extensively used in construction of a P. infestans genetic map. Multiple positive clones were identified for each AFLP marker tested. The pools were used to construct a contig of 11 BAC clones in a region of the P. infestans genome containing a cluster of three avirulence genes. The BAC contig is predicted to encompass the Avr11 locus but mapping of the BAC ends will be required to determine if the Avr3 and Avr10 loci are also present in the BAC contig. These results are an important step towards the positional cloning of avirulence genes from P. infestans, and the BAC library represents a valuable resource for largescale studies of oomycete genome organisation and gene content.

Chromosomes, Artificial, Bacterial↗
PubMed1994

Recombinations in individuals homozygous by descent localize the Friedreich ataxia locus in a cloned 450-kb interval.

The locus for Friedreich ataxia (FRDA), a severe neurodegenerative disease, is tightly linked to markers D9S5 and D9S15, and analysis of rare recombination events has suggested the order cen-FRDA-D9S5-D9S15-qter. We report here the construction of a YAC contig extending 800 kb centromeric to D9S5 and the isolation of five new microsatellite markers from this region. In order to map these markers with respect to the FRDA locus, all within a 1-cM confidence interval, we sought to increase the genetic information of available FRDA families by considering homozygosity by descent and association with founder haplotypes in isolated populations. This approach allowed us to identify one phase-known recombination and one probable historic recombination on haplotypes from Réunion Island patients, both of which place three of the five markers proximal to FRDA. This represents the first identification of close FRDA flanking markers on the centromeric side. The two other markers allowed us to narrow the breakpoint of a previously identified distal recombination that is > 180 kb from D9S5 (26P). Taken together, the results place the FRDA locus in a 450-kb interval, which is small enough for direct search of candidate genes. A detailed rare cutter restriction map and a cosmid contig covering this interval were constructed and should facilitate the search of genes in this region.

Base Sequence↗
PubMed2001

Physical and transcriptional map of the hereditary inclusion body myopathy locus on chromosome 9p12-p13.

Hereditary inclusion body myopathy (HIBM) is a group of neuromuscular disorders characterised by adult-onset, slowly progressive distal and proximal muscle weakness and typical muscle pathology. Previously, we have mapped the gene responsible for a recessive form of HIBM to chromosome 9p1 and narrowed the interval to one single YAC clone of 1 Mb in size. As a further step towards the identification of the HIBM gene, we have constructed a detailed physical and transcriptional map of this region. A high resolution BAC contig that includes the HIBM critical region, flanked by marker 327GT4 and D9S1859, was constructed. This contig allowed the precise localisation of 25 genes and ESTs to the proximal region of chromosome 9. The expression pattern of those mapped genes and ESTs was established by Northern blot analysis. In the process of refining the HIBM interval, 13 new polymorphic markers were identified, of which 11 are CA-repeats, and two are single nucleotide polymorphisms. Certainly, this map provides an important integration of physical and transcriptional information corresponding to chromosome 9p12-p13, which is expected to facilitate the cloning and identification not only of the HIBM gene, but also other disease genes which map to this region.

Chromosomes, Artificial, Bacterial↗
PubMed2001

Uniform integration of genome mapping data using intersection graphs.

MOTIVATION: The methods for analyzing overlap data are distinct from those for analyzing probe data, making integration of the two forms awkward. Conversion of overlap data to probe-like data elements would facilitate comparison and uniform integration of overlap data and probe data using software developed for analysis of STS data. RESULTS: We show that overlap data can be effectively converted to probe-like data elements by extracting maximal sets of mutually overlapping clones. We call these sets virtual probes, since each set determines a site in the genome corresponding to the region which is common among the clones of the set. Finding the virtual probes is equivalent to finding the maximal cliques of a graph. We modify a known maximal-clique algorithm such that it finds all virtual probes in a large dataset within minutes. We illustrate the algorithm by converting fingerprint and Alu-PCR overlap data to virtual probes. The virtual probes are then analyzed using double-linkage intersection graphs and structure graphs to show that methods designed for STS data are also applicable to overlap data represented as virtual probes. Next we show that virtual probes can produce a uniform integration of different kinds of mapping data, in particular STS probe data and fingerprint and Alu-PCR overlap data. The integrated virtual probes produce longer double-linkage contigs than STS probes alone, and in conjunction with structure graphs they facilitate the identification and elimination of anomalies. Thus, the virtual-probe technique provides: (i) a new way to examine overlap data; (ii) a basis on which to compare overlap data and probe data using the same systems and standards; and (iii) a unique and useful way to uniformly integrate overlap data with probe data.

Algorithms↗
PubMed1999

Identification and mapping of novel tumor suppressor loci on 6p in diffuse large B-cell non-Hodgkin's lymphoma.

Allelic deletions have been thought to be indicators of the presence of tumor suppressor genes (TSGs). As indicated by this allelotype study using 39 highly informative microsatellite markers distributed among all autosomal chromosomes, frequent loss of heterozygosity (LOH) has been found at 6p in B-cell non-Hodgkin's lymphoma. To identify the commonly deleted regions (CDRs), we performed fine deletion mapping using 26 highly polymorphic microsatellite markers on 6p. The most frequent LOH occurred at D651721, where 9 of 18 of the informative cases (50%) had allelic losses. Seventeen of 32 cases (53%) exhibited LOH at least at one locus on 6p. Ten of these 17 cases showed interstitial deletions, and their LOH patterns indicated two CDRs on 6p; one between D6S1721 and D6S260 (at 6p23-24), and the other between D6S265 and D6S291 (at 6p21). The genetic distance of both CDRs was 6 cM. The CDKN1A (p21) gene is reported to be located within the interval of the CDR at 6p21, but no mutation of the gene was found in these 32 patients. These data suggested that these two loci might harbor novel putative TSGs responsible for the pathogenesis of malignant lymphoma. We have constructed a contig of yeast artificial chromosome (YAC) clones spanning the most frequent CDR at 6p23-24. This YAC contig can be used for fine physical mapping of the region and cloning of candidate TSGs.

Adolescent↗
PubMed2000

Locus-specific contig assembly in highly-duplicated genomes, using the BAC-RF method.

Polyploidy, the presence of multiple sets of chromosomes that are similar but not identical, complicates both chromosome walking and assembly of sequence-ready contigs for many plant taxa including a large number of economically-significant crops. Traditional 'dot-blot hybridization' or PCR-based assays for identifying BAC clones corresponding to a mapped DNA landmark usually do not provide sufficient information to distinguish between allelic and non-allelic loci. A restriction fragment matching method using pools of BAC DNA in combination with dot-blots reveals the locus specificity of individual BACs that correspond to multi-locus DNA probes, in a manner that can efficiently be applied on a large scale. This approach also provides an alternative means of mapping DNA loci that exploits many advantages of 'radiation hybrid' mapping in taxa for which such hybrids are not available. The BAC-RF method is a practical and reliable approach for using high-density RFLP maps to anchor sequence-ready BAC contigs in highly-duplicated genomes, provides an alternative to high-density robotic gridding for screening BAC libraries when the necessary equipment is not available, and permits the expedient isolation of individual members of multigene or repetitive DNA families for a wide range of genetic and evolutionary investigations.

Chromosomes, Bacterial↗
PubMed2003

Identification and characterization of human FMNL1, FMNL2 and FMNL3 genes in silico.

FMNL (NM_005892.2) is a 5'-truncated partial cDNA encoding a Formin-homology protein related to DAAM1, DAAM2, DIAPH1 and DIAPH2. Here, we identified three members of FMNL gene family in the human genome by using bioinformatics. FMNL1 gene, corresponding to 5'-truncated KW-13 and FMNL cDNAs, was located within reference genomic contig NT_010748.9 (nucleotide position 100576-125849, forward orientation). FMNL2 gene, corresponding to KIAA1902 and FHOD2 cDNAs, was located within NT_005151.10 (nucleotide position 122465-436828, forward orientation). FMNL3 gene, corresponding to 5'-truncated DKFZp762B245 and KIAA2014 cDNAs, was located within NT_026397.10 (nucleotide position 209769-279037, reverse orientation). FMNL1, FMNL2 and FMNL3 genes encode A and B isoforms with the C-terminal divergence due to alternative splicing (cassette splicing of exon 26). FMNL1A (1100 aa), FMNL1B (1114 aa), FMNL2A (1087 aa), FMNL2B (1093 aa), FMNL3A (1028 aa) and FMNL3B (1027 aa) consist of FDD, FH1 and FH2 domains. Total amino-acid identity were as follows: FMNL1A vs. FMNL2A, 59.3%; FMNL1A vs. FMNL3A, 56.1%; FMNL2A vs. FMNL3A, 68.6%. FMNL1 gene was mapped to human chromosome 17q21. FMNL2 gene was linked to FNBP3/HYPA gene on chromosome 2q23.3, while FMNL3 gene was linked to FNBP3L/HYPC gene on chromosome 12q13. FMNL1 mRNA was expressed in natural killer cells, Burkitt lymphoma, pancreatic cancer, prostate cancer, and lung large cell carcinoma, FMNL2 mRNA in several normal tissues, diffuse-type gastric cancer, breast cancer, chondrosarcoma, melanoma, and glioblastoma, and FMNL3 mRNA in gastric cancer. FMNL1, FMNL2 and FMNL3 might be implicated in polarity control, invasion, migration, or metastasis through regulation of the Rho-related signaling pathway.

Alternative Splicing↗
PubMed2000

Analysis of 5S rDNA arrays in Arabidopsis thaliana: physical mapping and chromosome-specific polymorphisms.

A physical map of a pericentromeric region of chromosome 5 containing a 5S rDNA locus and spanning approximately 1000 kb was established using the CIC YAC clones. Three 5S rDNA arrays were resolved in this YAC contig by PFGE analysis and we have mapped different types of sequences between these three blocks. 5S rDNA units from each of these three arrays of chromosome 5, and from chromosomes 3 and 4, were isolated by PCR. A total of 38 new DNA sequences were obtained. Two types of 5S rDNA repeated units exist: the major variant with 0.5-kb repeats and one with short repeats (251 bp) only detected on YAC 11A3 from chromosome 3. Although the 38 sequences displayed noticeable heterogeneity, we were able to group them according to their 5S array origin. The presence of 5S array-specific variants was confirmed with the restriction polymorphism study of all the YACs carrying 5S units.

Animals↗
PubMed2005

A fine physical map of the rice chromosome 5.

A fine physical map of the rice (Oryza sativa spp. Japonica var. Nipponbare) chromosome 5 with bacterial artificial chromosome (BAC) and PI-derived artificial chromosome (PAC) clones was constructed through integration of 280 sequenced BAC/PAC clones and 232 sequence tagged site/expressed sequence tag markers with the use of fingerprinted contig data of the Nipponbare genome. This map consists of five contigs covering 99% of the estimated chromosome size (30.08 Mb). The four physical gaps were estimated at 30 and 20 kb for gaps 1-3 and gap 4, respectively. We have submitted 42.2-Mb sequences with 29.8 Mb of nonoverlapping sequences to public databases. BAC clones corresponding to telomere and centromere regions were confirmed by BAC-fluorescence in situ hybridization (FISH) on a pachytene chromosome. The genetically centromeric region at 54.6 cM was covered by a minimum tiling path spanning 2.1 Mb with no physical gaps. The precise position of the centromere was revealed by using three overlapping BAC/PACs for approximately 150 kb. In addition, FISH results revealed uneven chromatin condensation around the centromeric region at the pachytene stage. This map is of use for positional cloning and further characterization of the rice functional genomics.

Centromere↗
PubMed1998

Physical map of the human chromosome 8p12-p21 encompassing tumor suppressor and Werner's syndrome gene loci.

Detailed physical maps of the human genome are important resources for identification and isolation of genes responsible for diseases and for the study of their structure and function. We constructed a 2.0-Mb high-resolution physical map within the human chromosome 8p12-p21 region extending from marker D8S131 to D8S283. The map comprises a series of contigs mostly P1/PAC clones, which span the loci of potential tumor suppressor genes and the Werner's syndrome gene. Each P1/PAC DNA was defined by its size, restriction sites, terminal sequences, intermarker distances and location relative to major genes and markers. The genes on these P1/PAC DNAs were analyzed by an exon amplification method to determine their locations. The genes newly found by the exon amplification method together with other known genes, including those of glutathion reductase, a general transcription factor, protein phosphatase 2A beta subunit and Werner's syndrome, were precisely mapped within the contigs. These P1/PAC DNAs are useful reagents for the generation of new microsatellite markers to narrow the candidate region of the tumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in this region by linkage analysis.

Animals↗
PubMedDate not supplied

Refined subchromosomal location of 21 expressed sequence tags from unknown genes at region 11p15.

Twenty-one expressed sequence tags (ESTs) belonging to unidentified transcripts were mapped to 9 intervals at 11p 15.5-p15.3. Thirteen were mapped to a region of an estimated size of 7 Mb with the help of a YAC contig. The remaining eight were mapped outside this region, and the centromeric or telomeric position of the ESTs relative to the YAC contig was defined with the help of a cellular hybrid containing a derivative chromosome 11 with a break-point within the region covered by the contig. The average size of the intervals, where the ESTs were mapped, was estimated to be 0.3-0.4 Mb. The refinement of the chromosomal location of these ESTs could be of great help in the identification of potential candidate genes for disease locus mapping at 11p15.

Animals↗
PubMed1995

A YAC contig spanning the dominant retinitis pigmentosa locus (RP9) on chromosome 7p.

The dominant retinitis pigmentosa locus RP9 has previously been localized to 7p13-p15, in the interval D7S526-D7S484. We now report refinement of the locus to the interval D7S795-D7S484 and a YAC contig of approximately 4.8 Mb spanning this region and extending both distally and proximally from it. The contig was constructed by STS content mapping and physically orders 29 STSs in 28 YAC clones. The order of polymorphic markers in the contig is consistent with a genetic map that has been assembled using haplotype data from the CEPH pedigrees. This contig will provide a primary resource for the construction of a transcriptional map of this region and for the identification of the defective gene causing this form of adRP.

Base Sequence↗